2.9 Aortic ring assays
To establish the direct action of FGF2 and PEG-FGF2 conjugates on
vascular tissue, the thoracic aortae of 8-week-old mice from each group
were isolated surgically, cleaned, and dissected into 0.5 mm rings.
Rings were then embedded in 1 mg/mL of type I collagen (Millipore,
Cat#08-115) in a 96-well plate as described previously (Baker et
al. , 2011). The embedded rings were cultured in HG (33 mM) serum-free
endothelial basal medium (EBM) (Lonza, Cat#CC-3121) in the presence of
FGF2 or PEG-FGF2 conjugates. During the exponential growth phase,
angiogenic response data were obtained by counting the endothelial
microvessel sprouts growing out from the cultured rings. Before the
regression phase, rings were fixed with 4% (w/v) paraformaldehyde for
30 min. Pictures were taken on day 12 with a computer assisted
microscope (Eclipse Ni, NIKON), and the total number of branches was
counted using ImageJ (National Institutes of Health, USA).